EP1553102A1 - Synthèse d'oligonucléotides - Google Patents

Synthèse d'oligonucléotides Download PDF

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EP1553102A1
EP1553102A1 EP05075379A EP05075379A EP1553102A1 EP 1553102 A1 EP1553102 A1 EP 1553102A1 EP 05075379 A EP05075379 A EP 05075379A EP 05075379 A EP05075379 A EP 05075379A EP 1553102 A1 EP1553102 A1 EP 1553102A1
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deprotection
nucleoside
carbonate
group
synthesis
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English (en)
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Douglas J. Dellinger
Marvin H. Caruthers
Jason R. Betley
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Agilent Technologies Inc
University of Colorado
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Agilent Technologies Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates generally to nucleic acid chemistry and to the chemical synthesis of oligonucleotides. More particularly, the invention relates to an improved method for synthesizing oligonucleotides wherein carbonates are used as hydroxyl-protecting groups and "alpha-effect" nucleophiles such as peroxides are used in the deprotection thereof.
  • the invention has utility in the fields of biochemistry, molecular biology and pharmacology, and in medical diagnostic and screening technologies.
  • the chemical group conventionally used for the protection of nucleoside 5'-hydroxyls is dimethoxytrityl ("DMT"), which is removable with acid.
  • DMT dimethoxytrityl
  • This acid-labile protecting group provides a number of advantages for working with both nucleosides and oligonucleotides.
  • the DMT group can be introduced onto a nucleoside regioselectively and in high yield. E.I. Brown et al . (1979) Methods in Enzymol . 68 :109.
  • the lipophilicity of the DMT group greatly increases the solubility of nucleosides in organic solvents, and the carbocation resulting from acidic deprotection gives a strong chromophore, which can be used to indirectly monitor coupling efficiency.
  • the hydrophobicity of the group can be used to aid separation on reverse-phase HPLC. C. Becker et al . (1985) J. Chromatogr. 326 :219.
  • the N-6-benzoyl-protected deoxyadenosine nucleotide is especially susceptible to glycosidic cleavage, resulting in a substantially reduced yield of the final oligonucleotide.
  • the invention is thus addressed to the aforementioned deficiencies in the art, and provides a novel method for synthesizing oligonucleotides, wherein the method has numerous advantages relative to prior methods such as those discussed above.
  • the novel method involves the use of neutral or mildly basic conditions to remove hydroxyl-protecting groups, such that acid-induced depurination is avoided.
  • the reagents used provide for irreversible deprotection, significantly reducing the likelihood of unwanted side reactions and increasing the overall yield of the desired product.
  • the method provides for simultaneous oxidation of the intemucleoside phosphite triester linkage and removal of the hydroxyl-protecting group, eliminating the extra step present in conventional processes for synthesizing oligonucleotides; the method also avoids the extra step of removing exocyclic amine protecting groups, as the reagents used for hydroxyl group deprotection substantially remove exocyclic amine protecting groups.
  • the method can be used in connection with fluorescent or other readily detectable protecting groups, enabling monitoring of individual reaction steps. Further, the method is useful in carrying out either 3'-to-5' synthesis or 5'-to-3' synthesis.
  • the method readily lends itself to the highly parallel, microscale synthesis of oligonucleotides.
  • Still a further object of the invention is to provide such a method in which individual oligonucleotides are synthesized within the context of a highly dense, substantially parallel oligonucleotide array on a substrate surface.
  • Still an additional object of the invention is to provide nucleoside reagents useful in conjunction with the novel synthetic methods.
  • the invention is premised on the discovery that rapid and selective removal of suitable 5'-OH or 3'-OH protecting groups following phosphoramidite condensation can be achieved by employing nucleophiles, and particularly peroxy anions, that exhibit an "alpha effect" under neutral or mildly basic conditions. Further, it has now been discovered that rapid and selective deprotection can be achieved under such conditions by employing carbonate groups for 5'-OH or 3'-OH protection. Deprotection of nucleoside carbonates using peroxy anions can be conducted in aqueous solution, at neutral or mild pH, resulting in quantitative removal of the carbonate group and concomitant and quantitative oxidation of the internucleotide phosphite triester bond. Oligonucleotides synthesized using the novel methodology can be isolated in high yield and substantially free of detectable nucleoside modifications.
  • alpha effect as in an "alpha effect" nucleophilic deprotection reagent, is used to refer to an enhancement of nucleophilicity that is found when the atom adjacent a nucleophilic site bears a lone pair of electrons.
  • a nucleophile is said to exhibit an "alpha effect” if it displays a positive deviation from a Br ⁇ nsted-type nucleophilicity plot.
  • the invention features a method, in an oligonucleotide synthesis, for removing a protecting group from a protected nucleoside, by reacting the protected nucleoside or protected nucleotide with a nucleophile that exhibits an alpha effect at conditions of mildly basic pH, and particularly at conditions of pH 10 or less.
  • the invention provides for efficient solid-phase synthesis of oligonucleotides of lengths up to 25 nucleotides and greater.
  • Treatment using an alpha effect nucleophile according to the invention for removal of carbonate protecting groups is irreversible, resulting in breakdown of the carbonate and formation of CO 2 .
  • the method of the invention obviates the need for a separate oxidation step and a post-synthesis deprotection step to remove any exocyclic amine protecting groups that may be used.
  • the invention features a method for making an oligonucleotide array made up of array features each presenting a specified oligonucleotide sequence at an address on an array substrate, by first treating the array substrate to protect the hydroxyl moieties on the derivatized surface from reaction with phosphoramidites, then carrying out the steps of (a) applying droplets of an alpha effect nucleophile to effect deprotection of hydroxyl moieties at selected addresses, and (b) flooding the array substrate with a medium containing a selected protected phosphoramidite to permit coupling of the selected phosphoramidite onto the deprotected hydroxyl moieties at the selected addresses, and repeating the steps (a) and (b) to initiate and to sequentially build up oligonucleotides having the desired sequences at the selected addresses to complete the array features.
  • the droplets applied may comprise the protected phosphoramidite, and the alpha effect nucleophile may be used to flood
  • the deprotection reagents are aqueous, allowing for good droplet formation on a wide variety of array substrate surfaces. Moreover, because the selection of features employs aqueous media, small-scale discrete droplet application onto specified array addresses can be carried out by adaptation of techniques for reproducible fine droplet deposition from printing technologies. Further, as noted above, the synthesis reaction provides irreversible deprotection resulting in evolution of CO 2 , and thus quantitative removal of protecting groups within each droplet may be achieved.
  • the phosphoramidite reactions are carried out in bulk, employing an excess of the phosphoramidite during the coupling step (b), allowing for exclusion of water by action of the excess phosphoramidite as a desiccant.
  • a method for synthesizing oligonucleotides which comprises (a) condensing the 3' -OH or 5' -OH group of a support-bound nucleoside or oligonucleotide with a monomeric nucleoside phosphoramidite having a carbonate-protected hydroxyl group, to provide an intermediate in which the support-bound nucleoside or oligonucleotide is bound to the monomeric nucleoside through a phosphite triester linkage, and (b) treating the intermediate provided in step (a) with a deprotecting reagent effective to convert the carbonate-protected hydroxyl group to a free hydroxyl moiety and simultaneously oxidize the phosphite triester linkage to give a phosphotriester linkage.
  • (a) and (b) are carried out in aqueous solution.
  • step (b) is an irreversible reaction.
  • step (b) is conducted at neutral to mildly basic pH, in which case it is preferred that the deprotecting reagent used in step (b) comprises a nucleophile that exhibits an alpha effect at neutral to mildly basic pH.
  • Step (b) may be conducted at a pH of less than about 10.
  • the nucleophile is a peroxide, and preferably the peroxide is an inorganic peroxide of the formula M + OOH - , wherein M + is a counterion.
  • the counterion may be selected from the group consisting of H + , Li + , Na + , K + , Rb + and Cs + .
  • the peroxide is an organic peroxide of the formula ROOH, wherein R is hydrocarbyl optionally substituted with one or more nonhydrocarbyl substituents and optionally containing one or more nonhydrocarbyl linkages.
  • the peroxide may have the structure of formula (V), (VI) or (VII) (VII) R 10 ⁇ O ⁇ O - in which R 4 through R 10 are hydrocarbyl optionally substituted with one or more nonhydrocarbyl substituents and optionally containing one or more nonhydrocarbyl linkages.
  • the peroxide is one of t-butyl hydroperoxide, m -chloroperoxybenzoic acid, and mixtures thereof.
  • step (b) is conducted at a pH at which said nucleophile exhibits a maximum alpha effect, and preferably is conducted at a pH that is greater than the pKa of said nucleophile. Steps (a) and (b) may be repeated until an oligonucleotide having a desired sequence and length is provided.
  • step (a) involves condensation at the 5'-OH of the support-bound nucleoside or oligonucleotide.
  • the monomeric nucleoside phosphoramidite may have the structure of formula (II) wherein:
  • X is di(lower alkyl)amino or morpholino and Y is selected from the group consisting of lower alkyl, electron-withdrawing ⁇ -substituted aliphatic, electron-withdrawing substituted phenyl, and electron-withdrawing substituted phenylethyl. More preferably, Y is selected form the group consisting of methyl, ⁇ -cyanoethyl and 4-nitrophenylethyl.
  • R 1 is selected from the group consisting of o -nitrophenylcarbonyl, p -phenylazophenylcarbonyl, phenylcarbonyl, p -chlorophenylcarbonyl, 5'-( ⁇ -methyl-2-nitropiperonyl)oxycarbonyl ("MeNPOC”), 9-fluorenylmethylcarbonyl (“Fmoc”), 2,2,2-trichtoro-1,1-dimethylcarbonyl (“TCBOC”) and cyano-substituted lower alkyl carbonates.
  • R 3 comprises a moiety that becomes fluorescent or coloured upon cleavage of the carbonate -OCOOR 3 .
  • step (a) involves condensation at the 3'-OH of the support-bound nucleoside or oligonucleotide.
  • the monomeric nucleoside phosphoramidite preferably has the structure of formula (X) wherein:
  • X is preferably di(lower alkyl)amino or morpholino and Y is selected form the group consisting of lower alkyl, electron-withdrawing ⁇ -substituted aliphatic, electron-withdrawing substituted phenyl, and electron-withdrawing substituted phenylethyl.
  • Y is selected from the group consisting of methyl, ⁇ -cyanoethyl and 4-nitrophenylethyl.
  • R 1 is preferably selected from the group consisting of o -nitrophenylcarbonyl, p -phenylazophenylcarbonyl, phenylcarbonyl, p -chlorophenylcarbonyl, 5'-( ⁇ -methyl-2-nitropiperonyl)oxycarbonyl ("MeNPOC”), 9-fluorenylmethylcarbonyl ("Fmoc”), 2,2,2,-trichloro-1,1-dimethylarbonyl (“TCBOC”) and cyano-substituted lower alkyl carbonates.
  • R 3 preferably comprises a moiety that becomes fluorescent or coloured upon cleavage of the carbonate -OCOOR 3 .
  • Steps (i) and (ii) are preferably carried out in aqueous solution.
  • step (i) is an irreversible reaction.
  • Step (i) may be conducted at neutral to mildly basic pH;
  • Step (b) is preferably conducted at a pH of less than about 10.
  • the nucleophile is preferably a peroxide, and more preferably an inorganic peroxide of the formula M + OOH - , wherein M + is a counterion.
  • the counterion may be selected from the group consisting of H + , Li + , Na + , K + , Rb + and Cs + .
  • the peroxide may be an organic peroxide of the formula ROOH, wherein R is hydrocarbyl optionally substituted with one or more nonhydrocarbyl substituents and optionally containing one or more nonhydrocarbyl linkages.
  • the peroxide has the structure of formula (V), (VI) or (VII) (VII) R 10 ⁇ O ⁇ O - in which R 4 through R 10 are hydrocarbyl optionally substituted with one or more nonhydrocarbyl linkages.
  • the peroxide is one of t-butyl hydroperoxide, m -chloroperoxybenzoic acid, and mixtures thereof.
  • nucleoside monomer having the structure of formula (II) or formula (X) wherein:
  • X is di(lower alkyl)amino or morpholino and Y is selected from the group consisting of lower alkyl, electron-withdrawing ⁇ -substituted aliphatic, electron-withdrawing substituted phenyl, and electron-withdrawing substituted phenylethyl. More preferably, Y is selected from the group consisting of methyl ⁇ -cyanoethyl and 4-nitrophenylethyl.
  • R 3 is preferably selected from the group consisting of o -nitrophenylcarbonyl, p -phenylazophenylcarbonyl, phenylcarbonyl, p -chlorophenylcarbonyl, 5'-( ⁇ -methyl-2-nitropiperponyl)oxycarbonyl ("MeNPOC”), 9-fluorenylmethylcarbonyl ("Fmoc”), 2,2,2-trichloro-1,1-dimethylcarbonyl (“TCBOC”) and cyano-substituted lower alkyl carbonates.
  • R 3 comprises a moiety that becomes fluorescent or coloured upon cleavage of the carbonate -OCOOR 3 .
  • a particularly preferred nucleoside monomer is that having the structure of formula (II) or the structure of formula (X).
  • a kit for synthesizing an oligonucleotide on a solid support comprising a hydroxyl-derivatized support surface, a protecting group for protecting hydroxyl moieties on said derivatized support surface, at least one protected nucleoside, at least one nucleoside phosphoramidite, a nucleophile that exhibits an alpha effect at neutral to mildly basic pH, and reagents suitable for establishing pH and for carrying out reactions of deprotection, phosphoramidite coupling, and oxidation to form an internucleotide phosphotriester linkage.
  • nucleoside and nucleotide will include those moieties which contain not only the known purine and pyrimidine bases, but also modified purine and pyrimidine bases and other heterocyclic bases which have been modified (these moieties are sometimes referred to herein, collectively, as “purine and pyrimidine bases and analogs thereof”). Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, and the like.
  • protecting group as used herein is meant a species which prevents a segment of a molecule from undergoing a specific chemical reaction, but which is removable from the molecule following completion of that reaction. This is in contrast to a “capping group,” which permanently binds to a segment of a molecule to prevent any further chemical transformation of that segment.
  • Optional or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.
  • the phrase “optionally substituted” means that a non-hydrogen substituent may or may not be present, and, thus, the description includes structures wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.
  • the invention pertains to a method for synthesizing an oligonucleotide on a solid support, wherein a carbonate is used as a hydroxyl-protecting group and an alpha effect nucleophile is used to bring about deprotection.
  • the novel synthesis is based on a simple, two-step method of (1) coupling a hydroxyl-protected nucleoside monomer to a growing oligonucleotide chain, and (2) deprotecting the product, under neutral or mildly basic conditions, using an alpha effect nucleophilic reagent that also oxidizes the internucleotide linkage to give a phosphotriester bond.
  • the coupling and deprotection/oxidation steps are repeated as necessary to give an oligonucleotide having a desired sequence and length.
  • an unprotected nucleoside is covalently attached to a solid support to serve as the starting point for oligonucleotide synthesis.
  • the nucleoside may be bound to the support through its 3'-hydroxyl group or its 5'-hydroxyl group, but is typically bound through the 3'-hydroxyl group.
  • a second nucleoside monomer is then coupled to the free hydroxyl group of the support-bound initial monomer, wherein for 3'-to-5' oligonucleotide synthesis, the second nucleoside monomer has a phosphorus derivative such as a phosphoramidite at the 3' position and a carbonate protecting group at the 5' position, and alternatively, for 5'-to-3' oligonucleotide synthesis, the second nucleoside monomer has a phosphorus derivative at the 5' position and a carbonate protecting group at the 3' position.
  • This coupling reaction gives rise to a newly formed phosphite triester bond between the initial nucleoside monomer and the added monomer, with the carbonate-protected hydroxyl group intact.
  • the carbonate group is removed with an alpha effect nucleophile that also serves to oxidize the phosphite triester linkage to the desired phosphotriester.
  • a support-bound nucleoside monomer having the structure (I) wherein:
  • the protected monomer to be added has the structure of formula (II) in which B and R are as defined above with respect to the support-bound nucleoside of structural formula ( I ), and R 1 is COOR 3 , such that a carbonate group -OCOOR 3 is present at the 5' position.
  • R 3 is generally substituted or unsubstituted hydrocarbyl, including alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, optionally containing one or more nonhydrocarbyl linkages such as ether linkages, thioether linkages, oxo linkages, amine and imine linkages, and optionally substituted on one or more available carbon atoms with a nonhydrocarbyl substituent such as cyano, nitro, halo, or the like.
  • Preferred carbonate groups -OCOOR 3 are aryl carbonates, i.e., R 3 is aryl.
  • Suitable aryl carbonates include, for example, o -nitrophenylcarbonyl, p -phenylazophenylcarbonyl, phenylcarbonyl, p -chlorophenylcarbonyl, 5'-( ⁇ -methyl-2-nitropiperonyl)oxycarbonyl (“MeNPOC”), and 9-fluorenylmethylcarbonyl (“Fmoc”).
  • aryl carbonates have the structure Ar-L-O-(CO)-O- wherein Ar is an aromatic moiety, typically a monocyclic aromatic moiety such as a phenyl group, optionally substituted with one or more electron-withdrawing substituents such as halo, nitro, cyano, or the like, and L is a lower alkylene linkage.
  • Preferred alkyl carbonate substituents are fluorinated alkyl carbonates such as 2,2,2-trichloro-1,1-dimethylcarbonyl ("TCBOC”) and cyano-substituted alkyl carbonates such as 1,1-dimethyl-2-cyanoethyl carbonate
  • R 3 may also be a fluorescent or colored moiety.
  • R 3 becomes fluorescent or colored upon cleavage of the carbonate -OCOOR 3 , but is neither fluorescent nor colored when bound to the nucleoside in carbonate form.
  • the reaction may be monitored by detecting a fluorescent or colored cleavage product.
  • R 3 may be fluorescent or colored when bound to the nucleoside in carbonate form, such that the presence of the newly attached monomer can be immediately detected.
  • fluorescent and colorimetric species examples include, but are not limited to: xanthenes such as fluoresceins, eosins and erythrosins, with preferred fluorescein compounds exemplified by 6-carboxy-fluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-4',5'-dichloro-5- and 6-carboxyfluorescein, 2',7'-dimethoxy-4',5'-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1',2',7',8'-dibenzo-5- and 6-carboxy-4,7-dichlorofluorescein, 2',7'-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1'
  • R 2 is a phosphorus derivative that enables coupling to a free hydroxyl group.
  • Preferred phosphorus derivatives are phosphoramidites, such that R 2 has the structure (III) wherein X is NQ 1 Q 2 in which Q 1 and Q 2 may be the same or different and are typically selected from the group consisting of alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, optionally containing one or more nonhydrocarbyl linkages such as ether linkages, thioether linkages, oxo linkages, amine and imine linkages, and optionally substituted on one or more available carbon atoms with a nonhydrocarbyl substituent such as cyano, nitro, halo, or the like.
  • Q 1 and Q 2 represent lower alkyl, more preferably sterically hindered lower alkyls such as isopropyl, t -butyl, isobutyl, sec -butyl, neopentyl, tert-pentyl, isopentyl, sec-pentyl, and the like. Most preferably, Q 1 and Q 2 both represent isopropyl. Alternatively, Q 1 and Q 2 may be linked to form a mono- or polyheterocyclic ring having a total of from 1 to 3, usually 1 to 2 heteroatoms and from 1 to 3 rings.
  • Q 1 and Q 2 together with the nitrogen atom to which they are attached represent, for example, pyrrolidone, morpholino or piperidino.
  • Q 1 and Q 2 have a total of from 2 to 12 carbon atoms.
  • Examples of specific -NQ 1 Q 2 moieties thus include, but are not limited to, dimethylamine, diethylamine, diisopropylamine, dibutylamine, methylpropylamine, methylhexylamine, methylcyclopropylamine, ethylcyclohexylamine, methylbenzylamine, methylcyclohexylmethylamine, butylcyclohexylamine, morpholine, thiomorpholine, pyrrolidine, piperidine, 2,6-dimethylpiperidine, piperazine, and the like.
  • Y is hydrido or hydrocarbyl, typically alkyl, alkenyl, aryl, aralkyl, or cycloalkyl.
  • Y represents: lower alkyl; electron-withdrawing ⁇ -substituted aliphatic, particularly electron-withdrawing ⁇ -substituted ethyl such as ⁇ -trihalomethyl ethyl, ⁇ -cyanoethyl, ⁇ -sulfoethyl, ⁇ -nitro-substituted ethyl, and the like; electron-withdrawing substituted phenyl, particularly halo-, sulfo-, cyano- or nitro-substituted phenyl; or electron-withdrawing substituted phenylethyl.
  • Y represents methyl, ⁇ -cyanoethyl, or 4-nitrophenylethyl.
  • the coupling reaction is conducted under standard conditions used for the synthesis of oligonucleotides and conventionally employed with automated oligonucleotide synthesizers.
  • the product (IV) is treated with an "alpha effect" nucleophile in order to remove the carbonate protecting group at the 5' terminus, thus converting the moiety -OR' to -OH.
  • the alpha effect nucleophile also oxidizes the newly formed phosphite triester linkage -O-P(OY)-O- to give the desired phosphotriester linkage
  • this step is conducted in an aqueous solution at neutral pH or at a mildly basic pH, depending on the pKa of the nucleophilic deprotection reagent.
  • the pH at which the deprotection reaction is conducted must be above the pKa of the deprotection reagent for the reagent to be effective.
  • the reaction is conducted at a pH of less than about 10.
  • the nucleophilic deprotection reagent that exhibits an alpha effect is a peroxide or a mixture of peroxides, and the pH at which deprotection is conducted is at or above the pKa for formation of the corresponding peroxy anion.
  • the peroxide may be either inorganic or organic. Suitable inorganic peroxides include those of the formula M + OOH - , where M is any counteranion, including for example H + , Li + , Na + , K + , Rb + , Cs + , or the like; and lithium peroxide or hydrogen peroxide can be particularly suitable.
  • Suitable organic peroxides include those of the formula ROOH, where R is selected from the group consisting of alkyl, aryl, substituted alkyl and substituted aryl. More particularly, the organic peroxide will have one of the following three general structures (V), (VI) or (VII) (VII) R 10 ⁇ O ⁇ O - in which R 4 through R 10 are generally hydrocarbyl optionally substituted with one or more nonhydrocarbyl substituents and optionally containing one or more nonhydrocarbyl linkages.
  • R 4 through R 10 are independently selected from the group consisting of hydrido, alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl, alkynyl aralkynyl, cycloalkynyl, substituted aralkyl, substituted cycloalkyl, substituted cycloalkylalkyl, substituted alkenyl, substituted cycloalkeayl, substituted alkynyl substituted aralkynyl, substituted cycloalkynyl; t-butyl-hydroperoxide or metachloroperoxybenzoic acid can be particularly suitable.
  • the m -chloroperbenzoic acid (mCPBA) peroxy anion exhibits a strong alpha effect towards the p -chlorophenylcarbonate electrophile, and that, accordingly, the peroxyanion of mCPBA is a particularly effective deprotection reagent for removal of p -chlorophenylcarbonate protecting groups.
  • the product of this simultaneous deprotection and oxidation step may thus be represented as follows: wherein B, R and Y are as defined earlier herein.
  • This latter reaction also gives rise to the by-products R 3 O - and carbon dioxide, insofar as nucleophilic attack of the peroxide deprotection reagent cleaves the carbonate linkage as follows:
  • the use of a peroxy anion to effect simultaneous removal of the carbonate protecting group and oxidation of the internucleotide linkage also removes, to a large extent, exocyclic amine-rotecting groups such as acetyl, trifluoroacetyl, difluoroacetyl and trifluoroacetyl moieties.
  • an added advantage herein is the elimination of a separate post-synthetic reaction step to remove exocyclic amine-protecting groups, as is required with conventional methods of synthesizing oligonucleotides. Elimination of this additional step significantly decreases the time and complexity involved in oligonucleotide synthesis.
  • peroxy anions as deprotection reagents herein may be readily activated or inactivated by simply changing pH. That is, the effectiveness of peroxides as nucleophiles is determined by their pKa. In buffered solutions having a pH below the pKa of a particular peroxide, the peroxides are not ionized and thus are non-nucleophilic. To activate a peroxide and render it useful as a deprotection reagent for use herein, the pH is increased above the pKa so that the peroxide is converted to a nucleophilic peroxy anion. Thus, one can carefully control the timing and extent of the deprotection reaction by varying the pH of the peroxide solution used.
  • Figure 2 schematically illustrates 3'-to-5' synthesis of an oligonucleotide using the method of the present invention.
  • the moiety "Arco" represents the carbonate protecting group p -chlorophenylcarbonyl.
  • the synthesis may be contrasted with that schematically illustrated in Figure 1, the prior, conventional method employing DMT protection and separate oxidation and deprotection steps.
  • Figure 3A protection and deprotection of hydroxyl groups using DMT is a reversible process, with the DMT cation shown being a relatively stable species.
  • FIG. 3B illustrates the irreversible deprotection reaction of the present invention, wherein nucleophilic attack of the peroxy anion irreversibly cleaves the carbonate moiety, i.e., the O- p -chlorophenylcarbonyl group, giving rise to carbon dioxide and the p -chlorophenol anion.
  • the reaction is not "reversible,” insofar as there is no equilibrium reaction in which a cleaved protecting group could reattach to the hydroxyl moiety, as is the case with removal of DMT.
  • the method of the invention also lends itself to synthesis in the 5'-to-3' direction.
  • the initial step of the synthetic process involves attachment of a nucleoside monomer to a solid support at the 5' position, leaving the 3' position available for covalent binding of a subsequent monomer.
  • a support-bound nucleoside monomer is provided having the structure (IX) wherein O represents the solid support or a support-bound oligonucleotide chain, R is hydrido or hydroxyl, and B is a purine or pyrimidine base.
  • the protected monomer to be added has the structure of formula (X) wherein the carbonate protecting group is present at the 3' position, i.e., R 1 is COOR 3 where R 3 is as defined previously, and R 2 represents a phosphorus derivative that enables coupling to a free hydroxyl group, preferably a phosphoramidite having the structure (III) wherein X and Y are as defined earlier herein.
  • the coupling reaction in which the nucleoside monomer becomes covalently attached to the 3' hydroxyl moiety of the support bound nucleoside is conducted under reaction conditions identical to those described for the 3'-to-5' synthesis. This step of the synthesis gives rise to the intermediate (XI)
  • the coupling reaction is followed by treatment of the product (XI) with an alpha effect nucleophile in order to remove the carbonate protecting group at the 3' terminus, thus converting the moiety -OR 1 to -OH, and to oxidize the internucleotide phosphite triester linkage to give the desired phosphotriester linkage.
  • the two-step process of coupling and deprotection/oxidation is repeated until the oligonucleotide having the desired sequence and length is obtained.
  • the oligonucleotide may, if desired, be cleaved from the solid support.
  • the synthetic methods of the invention may be conducted on any solid substrate having a surface to which chemical entities may bind.
  • Suitable solid supports are typically polymeric, and may have a variety of forms and compositions and derive from naturally occurring materials, naturally occurring materials that have been synthetically modified, or synthetic materials.
  • suitable support materials include, but are not limited to, polysaccharides such as agarose (e.g., that available commercially as Sepharose®, from Pharmacia) and dextran (e.g., those available commercially under the tradenames Sephadex® and Sephacyl®, also from Pharmacia), polyacrylamides, polystyrenes, polyvinyl alcohols, copolymers of hydroxyethyl methacrylate and methyl methacrylate, silicas, teflons, glasses, and the like.
  • polysaccharides such as agarose (e.g., that available commercially as Sepharose®, from Pharmacia) and dextran (e.g., those available commercially under the tradenames Sephadex® and Sephacyl®, also from Pharmacia), polyacrylamides, polystyrenes, polyvinyl alcohols, copolymers of hydroxyethyl methacrylate and methyl methacrylate, silicas, teflons, glasses
  • the initial monomer of the oligonucleotide to be synthesized on the substrate surface is typically bound to a linking moiety which is in turn bound to a surface hydrophilic group, e.g., to a surface hydroxyl moiety present on a silica substrate.
  • the invention features a method for making an oligonucleotide array made up of array features each presenting a specified oligonucleotide sequence at an address on an array substrate.
  • the array substrate is treated to protect the hydroxyl moieties on the derivatized surface from reaction with phosphoramidites or analogous phosphorus groups used in oligonucleotide synthesis. Protection involves conversion of free hydroxyl groups to -OR' groups, i.e., to carbonate-protected species.
  • the method then involves (a) applying droplets of an alpha effect nucleophile to effect deprotection of hydroxyl moieties at selected addresses and oxidation of the newly formed internucleotide phosphite triester linkages, followed by (b) flooding the array substrate with a medium containing a selected nucleoside monomer having the structure of either Formula (II) (for 3'-to-5' synthesis) or Formula (X) (for 5'-to-3' synthesis).
  • Step (a), deprotection/oxidation, and step (b), monomer addition are repeated to sequentially build oligonucleotides having the desired sequences at selected addresses to complete the array features.
  • the applied droplets may comprise the selected nucleoside monomer, while the alpha effect nucleophile is used to flood the array substrate; that is, steps (a) and (b) are essentially reversed.
  • the deprotection reagents are aqueous, allowing for good droplet formation on a wide variety of array substrate surfaces. Moreover, because the selection of features employs aqueous media, small-scale discrete droplet application onto specified array addresses can be carried out by adaptation of techniques for reproducible fine droplet deposition from printing technologies.
  • the invention additionally provides protected nucleoside monomers as novel compositions of matter useful, inter alia , in the synthesis of oligonucleotides as described herein.
  • the novel monomers have the structural formulae (II) and (X) wherein:
  • Reagent (II) used for 3'-to-5' synthesis, is readily prepared by reaction of the unprotected nucleoside with the haloformate R 3 O-(CO)-Hal wherein Hal represents halo, typically chloro, and R 3 is as defined previously, in the presence of a base effective to catalyze the nucleophilic reaction, e.g., pyridine.
  • This step results in a 5'-carbonate, as follows:
  • the intermediate so prepared is then phosphitylated with the phosphoramidite PX 2 (OY) wherein X and Y are as defined earlier, resulting in conversion of the 3'-hydroxyl moiety to the desired substituent -O-PX(PY), i.e., -OR 2 :
  • a specific example of this synthesis is illustrated schematically in Figure 4, wherein "Arco” represents the aryloxycarbonyl group p -chlorophenylcarbonyl, iPr represents isopropyl, and B is either N 6 -benzoyl-protected deoxyadenine, N 4 -Fmoc-protected deoxycytidine, N 2 -isobutyryl-protected deoxyguanine or thymine.
  • the unprotected base is reacted with 4-chlorophenyl chloroformate in the presence of pyridine to give the carbonate-protected 5'-OH, followed by phosphitylation using (iPr 2 N) 2 PO(CH 2 ) 2 CN, i.e., ⁇ -cyanoethyl-N,N-diisopropylamino phosphoramidite.
  • Reagent (X) used for 5'-to-3' synthesis, may be prepared by first synthesizing a 5'-protected nucleoside using a conventional 5'-OH protecting group such as DMT. This 5'-protected nucleoside is then reacted with the haloformate R 3 O-(CO)-Hal, which, as above, is done in the presence of a base effective to catalyze the nucleophilic reaction, e.g., pyridine.
  • a base effective to catalyze the nucleophilic reaction e.g., pyridine.
  • the syntheses were conducted generally as follows. Deoxythymidine (2 mmol) was co-evaporated with anhydrous pyridine (2 ⁇ 20 ml), then redissolved in dry pyridine (40 ml). The corresponding chloroformate (2.2 mmol) was added and the mixture stirred at room temperature (25°C) for 2 hr. The reaction was quenched with water (1 ml), then concentrated. The residual pyridine was removed by co-evaporation with toluene (40 ml).
  • the resulting gum was dissolved in CH 2 Cl 2 , extracted with 10% aqueous NaHCO 3 , and dried over Na 2 SO 4 . After concentration, the product was loaded onto a silica gel column (50 g) and eluted with CH 2 Cl 2 using a methanol gradient (0-3%). Product fractions were collected and concentrated to a foam.
  • the 3'-hydroxyl group of 5'-O-DMT-deoxythymidine was protected with a phenyloxycarbonyl (2 a ), a benzoyl (2 b ), and an acetyl (2 c ) group.
  • the stability of these 3'-protecting groups was determined by TLC using deprotection conditions C and D (Table 2). Under both these conditions, the phenyl carbonate was completely removed in less than 2 min.
  • the 3'-benzoyl group was completely stable under both conditions for 140 min.
  • the 3'-acetyl group was cleaved to a small extent (less than 3%) over the 140 min exposure to deprotection condition A (pH 10.0).
  • the 3-benzoyl group was completely stable for the 140 min exposure to condition B .
  • Oligonucleotides were synthesized on CPG using an automated DNA synthesizer (ABI model 380A).
  • the synthesis cycle used for 5'-DMT protected nucleoside phosphoramidites (Cycle 1) is shown in Table 4. This cycle was initially modified for the use of 5'-carbonate protected nucleoside phosphoramidites simply by substituting the alternative deprotection mixtures for the 3% TCA solution (Step 8, Table 4) and varying the exposure times. For the synthesis of longer sequences using 5-carbonate protected nucleoside phosphoramidites, it was necessary to separate the deprotection mixture into a two-component system (Table 3).
  • Table 4 shows the optimized cycle for synthesis using 5'-carbonate protected nucleoside phosphoramidites (Cycle 2): TWO-COMPONENT SYSTEM FOR STORAGE OF DEPROTECTION SOLUTION C Solution C-1 30 % H 2 O 2 (10 ml), LiOH (280 mg), dioxane (7.5 ml), 2.5 M Tris-Base (15 ml), water (42.5 ml) Solution C-2 50 - 60 % mCPBA (1.78 g), dioxane (42.5 ml) OLIGONUCLEOTIDE SYNTHESIS CYCLES Step # Function Reagent Cycle 1 Time, sec.
  • the oligonucleotides synthesized on the solid support were deprotected with concentrated ammonium hydroxide (55° C, 24 hr). The ammonium hydroxide solutions were removed from the support and evaporated to dryness. The crude oligonucleotides were reconstituted in distilled water and stored at -20° C.
  • HPLC analysis was performed by ion-exchange HPLC (Nucleogen 60-7DEAE, 4 mm ID ⁇ 125 mm). Oligonucleotides were eluted from the column with a LiCl gradient (0.0-0.7 M) in a water/acetonitrile (60/40, v/v) buffer containing sodium acetate (0.002 M, pH 6.0).
  • Thymidyl-3'-5'-deoxythymidine was synthesized on solid-support using 5'-O-dimethoxytrityl-3'-O-(2-cyanoethyl)-N,N-diisopropylaminodeoxythymidinephosphoramidite.
  • the dimer was elongated to a trimer using a 5'-O-aryloxycarbonyl-3'-O-(2-cyanoethyl)-N,N-diisopropylaminodeoxythymidinephosphoramidite and synthesis cycle 1 (Table 4).
  • Deprotection of the carbonate was then attempted using deprotection mixture C, at 1 min increments, from 1-15 min.
  • the extent of deprotection was determined by the yield of the subsequent coupling reaction using a standard 5'-DMT-dT phosphoramidite. Deprotection efficiency for the 5'-O-arylcarbonate was determined using ion-exchange HPLC. The percent deprotection was calculated by integration and normalization of peak areas for the corresponding trimers and tetramers, assuming quantitative coupling reactions. The optimum deprotection time and extent of deprotection for each aryloxycarbonyl group is summarized in Table 5.
  • oligothymidylate tetramers synthesized using the standard DMT protected phosphoramidites of thymidine. These tetramers were then analyzed using ion-exchange HPLC. There were no detectable differences in the yield or purity of any of the oligomers.
  • Oligothymidylate tetramers were then synthesized using this same synthesis cycle, which was again modified by the removal of the iodine oxidation step.
  • This concomitant deprotection and oxidation cycle produced tetramers of identical yield and purity to the standard DMT phosphoramidite synthesis.
  • Decomposition of MCPBA in the presence of LiOH results in the deprotection mixture being effective for only a few hours.
  • Oligonucleotide synthesis using 5'-O-arylcarbonate nucleoside phosphoramidites was carried out with and without acetic anhydride capping. No adverse effects on the yield of final product or increases in the appearance of n-1 products were observed in absence of capping. This is contrary to what is seen with the use of DMT protected phosphoramidites in the absence of capping. Anion-exchange HPLC profiles of crude synthesis products of oligothymidylate decamers were produced.
  • the unprotected heterocyclic bases cytosine and adenine are susceptible to N-oxidation by peracids and peroxides under stringent conditions, and oxidative reactions that result in ring cleavage of uracil, thymidine and guanosine in the presence of highly concentrated peroxides at elevated temperatures have been described.
  • 5'-O-DMT-protected nucleosides, N-protected with a (di-N-butylamino)methylene group were dissolved in deprotection mixture C and allowed to react for 24 hrs.
  • the tritylated nucleosides were extracted from the aqueous deprotection mixture with CHCl 3 and analyzed by 13 C NMR and TLC. Neither formation of N-oxides nor attack at the 5,6-double bond of thymidine (leading to ring cleavage) was detected.
  • This example demonstrates extension of the method of the invention to synthesis of mixed oligonucleotide sequences, employing substituted aryl carbonate protected phosphoramidite synthons, and following each coupling reaction by treatment with a mixture of peroxy-anions at mild pH (less than 10) to deprotect and concomitantly oxidize the internucleotide linkage.
  • the method is high-yielding, and effective for the four main 2'-deoxynucleotides. Synthesis in both the 3' - 5' direction and the 5' - 3' direction were carried out, with equal effect.
  • the protected nucleoside phosphoramidites were prepared as follows.
  • the 3'- or 5'-protected nucleoside (5.00 mmol) and tetrazole (175 mg, 2.50 mmol) were dried under vacuum for 24h and then dissolved in trichloromethane (100 mL).
  • 2-cyanoethyl- N , N , N' , N' -tetraisopropyl phosphane (2.06 mL, 6.50 mmol) was added in one portion and the mixture stirred over 1 hour. The reaction mixture was washed with sat.
  • the four 5'-aryloxycarbonyl-3'-nucleoside phosphoramidites were prepared by the straightforward two-step procedure shown generally in Figure 4.
  • a first step commercially available base protected 2'-oligodeoxynucleosides were selectively aryl carbonate protected at the 5' position by treatment with 4-chlorophenyl chloroformate in dilute anhydrous pyridine to yield 5'-aryloxycarbonyl protected compounds in moderate to good yield.
  • the use of more concentrated reaction mixtures resulted in an increase in the amounts of isolated 3'- and 3', 5'- bis -aryloxycarbonyl-protected materials.
  • the resulting compounds were phosphitylated using the method described in Barone et al., supra , to furnish high yields following column chromatography.
  • the deprotection mixture was formulated in two parts, which were mixed immediately prior to use.
  • Solution F 3.1% w/v lithium hydroxide monohydrate (10 mL), 1.5 M 2-amino-2-methyl-1-propanol pH 10.3 (15 mL), 1,4 dioxane (17.5 mL).
  • Solution G 1,4-dioxane (32.5 mL), 50 - 83% 3-chloroperbenzoic acid (1.78 g), 30% hydrogen peroxide (12 mL).
  • the initial pH of the deprotection mixture was 9.6 ⁇ 0.05.
  • the initial deprotection mixture was altered by varying the strength of the lithium hydroxide solution.
  • the HPLC obtained for 5'-CAGTTGTAAACGAGTT-3' is shown in Figure 6.
  • the calculated molecular weight for 5'-CAGTTGTAAACGAGTT-3' is 4921.1; the actual molecular weight determined using MALDI (Matrix Absorption Laser Desorption Ionization) TOF (Time of Flight) analysis was 4921.9.
  • the MALDI TOF spectrum is shown in Figure 7.
  • the stability of the standard base protecting groups A Bz , C Bz , and G ibu during exposure to the deprotection mixture was tested by incubating 5'-DMT base-protected deoxynucleosides at room temperature with a large excess of the deprotection mixture. The extent of cleavage of the base protecting groups over time was measured by TLC. The approximate T 1/2 values for A Bz , C Bz , and G ibu were approximately 1 ⁇ 2 hour, 2 hours, and 1 day, respectively, and unlikely to present difficulties for syntheses.

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US8309706B2 (en) 2012-11-13
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US6222030B1 (en) 2001-04-24
US20040230052A1 (en) 2004-11-18
US20060252924A1 (en) 2006-11-09
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US7101986B2 (en) 2006-09-05
EP0984021A3 (fr) 2001-08-01
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US7271258B2 (en) 2007-09-18
US20020045221A1 (en) 2002-04-18

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